Component 6 will focus on the role of one type of glutamate receptor, the N-methyl-D-aspartate receptor (NMDAR), in initial sensitivity to ethanol. Ethanol inhibits NMDAR function, and NMDARs play an important role in brain function. We have observed that long-Sleep (LS) and Short-Sleep (SS) mice, which differ in their initial sensitivity to the hypnotic effects of ethanol, show marked behavioral differences to the NMDAR antagonists MK- 801 and CPP. SS mice exhibited greater locomotor activation than LS mice at lower drug doses; and LS, but not SS, mice were sedated at higher drug doses. We have also found line differences in the density of [3H]MK-801 binding sites and ratio of NMDAR subunits (NR2A:NR2B) in LS and SS mouse brains. Therefore, this proposal focuses on the NMDAR as a candidate gene for differential ethanol sensitivity in the inbred lines of LS (ILS) and SS (ISS) mice. They hypothesis being tested is that: Differences between ILS/ISS in initial sensitivity to ethanol may be due to differences in NMDAR expression, composition, post-translational processing and/or signal transduction. Quantitative autoradiographic analyses of [3H]MK-801 and [3H]CPP binding and quantitative western analysis of NR1, NR2A, NR2B and NR2C subunits will be used to determine whether differences in NMDAR expression are localized to specific regions of the ILS and ISS mouse brains, particularly those involved in locomotor activity. In order to assess whether ILS and ISS mouse brain NMDARs contribute to the differential ethanol sensitivity of these mice, behavioral studies with MK-801 and CPP will be conducted using the LSxSS recombinant inbred strains. Next, in vivo electrochemical measures of NMDA-induced nitric oxide generation in intact brain and in vitro measures of NMDA induced electrophysiological activity in brain slices will be used to determine whether differences also exist in NMDAR signal transduction and in ethanol's inhibition of NMDAR activity in ILS and ISS mouse brain. Lastly, the contribution of potential differences in NMDAR subunit composition and/or phosphorylation state in specific regions of ILS and ISS brain will be investigated using co-immunoprecipitation assays, quantitative western analyses and front phosphorylation assays. The results of these experiments will increase our understanding of how excitatory glutamate systems contribute to genetic differences in initial sensitivity to ethanol. Understanding the factors mediating initial ethanol sensitivity is important because low initial sensitivity has been found to be associated with high risk for alcoholism.